Genetic variability in microbial eukaryotes reshapes marine biodiversity assessment in the age of amplicon sequencing

PLOS One | https://doi.org/10.1371/journal.pone.0326053 June 20, 2025 2 / 29 Introduction DNA sequencing has provided a wealth of information that has been transforming our understanding of marine microbial eukaryote species richness, biogeography, community composition, and evolutionary relationships [1,2]. In the field of ecology, high-throughput sequencing has enabled the detection of microorganisms that are oth- erwise too rare, fragile, fastidious, or morphologically indistinct to be readily observed with classical approaches of microscopy and culture. However, at this new frontier of DNA sequencing comes new challenges regarding data interpretation and integration with previous knowledge [3]. Metabarcoding is a highly-efficient technique for measur- ing biodiversity in environmental samples, which involves amplifying and sequencing a specific region within a chosen marker gene that is common to all targeted taxa (typically the 18S rRNA gene for microbial eukaryotes, a.k.a. “protists”). In this way, metabarcoding reveals the genetic richness of a sample, but ultimately the taxonomic assignment and interpretation of those gene sequences is limited by the quality of curated sequence databases (such as the Protist Ribosomal Reference Database, PR2 [4]). Due to the laborious nature of linking morphological and molecular data, these databases so far encompass only a small fraction of protistan richness, result- ing in many metabarcoding sequences with dubious taxonomic identities and little consensus as to what magnitude of sequence variation should delineate meaningful

biological units. Furthermore, while morphology-based taxonomy remains the gold standard in protistology, it is known to be imperfect, especially for rare, poorly- studied, and morphologically-indistinct taxa [3]. This poses the question: how can we best utilize genetic sequence information that is often only loosely tethered (if at all) to the morphological concepts that have historically been considered the common denominator in biological sciences? Discrepancies between morphology-based and sequence-based taxonomic assess- ments for some protistan groups can be confounding, and for one ubiquitous group of marine planktonic protists, the polycystine Radiolaria [5], these differences are partic- ularly stark. More than a century of taxonomic inventorying and description of polycys- tine radiolarian specimens in marine sediments, plankton nets, and sediment traps has yielded approximately 445 formal morphological species [6] and perhaps up to 600– 800, counting those that await formal description: most of these in orders Nassellaria and Spumellaria [5]. By contrast, the Tara Oceans expedition revealed an astounding ~6,600 polycystine genetic operational taxonomic units (OTUs) during a sampling expedition that targeted the sunlit ocean alone, and the vast majority of those OTUs were assigned to order Collodaria (another important polycystine group) rather than Nassellaria or Spumellaria [7]. This underscores how protistan morphological species descriptions cannot necessarily be expected to align with other modes of taxonomic

delineation, including concepts based on genetic similarity [8,9,10]. There is thus an urgent need to probe this mismatch between morphological and molecular richness so we can improve interpretations of the sequence datasets in genetic diversity surveys. Beyond epitomizing the disconnect between protistan morphological and molec- ular diversity, polycystine radiolarians are an ideal study system because their and reproduction in any medium, provided the original author and source are credited. Data availability statement: Sequence data underlying the analyses in this article are available from Zenodo (DOI: 10.5281/zeno- do.15550382). The raw, unprocessed sequence data are also available for download from the NCBI Sequence Read Archive (SRA) with sample accession numbers SAMN48798827 – SAMN48798999 (under BioProject PRJNA1269742). Funding: This project was support- ed by the National Science Foundation Postdoctoral Research Fellowship in Biology Award #2109767 (to ST) and the Simons Collaboration on Ocean Processes and Ecology p49802 (to DAC). MMS was supported by a postdoctoral fellowship from the Beatriu de Pinós programme of the Government of Catalonia's Secretariat for Universities and Research of the Ministry of Economy and Knowledge (grant #2021BP00068). Competing interests: The authors have declared that no competing interests exist.